SUMMARY Crowding and heterogeneity: Biomolecular organization in vivo is driven by crowding and heterogeneity. To date, protein structure, dynamics, and folding have been studied almost exclusively in simple buffer solutions, yet it is has recently become evident that most ?test tube? studies cannot be directly translated to cellular environments. Nonspecific electrostatic interactions, excluded volume effects, and disrupted hydrogen-bond networks dictate protein thermodynamics in these complex environments. While the prevailing view from these is that excluded-volume effects favor the more compact native states, our group, along with others, found that enthalpic contributions strengthen protein-water hydrogen bonds. These interactions can increase backbone exposure and consequently destabilize folded states. Thus, there is an immediate need to quantify interactions between biomolecules in accurate cell-like environments. The present studies are critical first step towards understanding protein structure and dynamics in vivo. Our project aims to characterize the structure, dynamics, and stability of proteins in crowded solutions that accurately mimic the cytoplasm. Specifically, we will quantify the degree of molecular heterogeneity and establish the role of macromolecular crowding on protein-protein and protein-water contacts. Protein-protein interactions and ion channel gating mechanisms: Calmodulin (CaM) regulates biological function by modulating the behavior of a wide range of proteins including many ion channels. CaM mutations or mutations within CaM-regulated ion channels are responsible for neurological and cardiovascular diseases. CaM can be considered a ?Ca-sensing domain? for multiple ion channels, but the dynamic association between CaM and ion channels make mechanistic studies challenging. The first complete structures of an ion channel with CaM were solved earlier this year (2018). These underscore the fact that the gating mechanisms remain incompletely understood. For example, eight states are required to model patch clamp measurements, but only two structures (open/closed) are known. We propose to investigate gating mechanisms through a detailed biophysical examination of dynamic CaM-channel interactions using a peptide that mimics the CaM binding domain of the SK2 channel (KCa2.2). SK channels are important in a wide variety of physiological systems and offer many advantages as a system for understanding Ca2+-CaM-mediated gating. If successful, our studies will produce a stepwise mechanistic view of CaM-mediated channel activation.